Chaperonin 10 immunosuppression

ABSTRACT

The invention is directed to the use of cpnl0 in transplantation and particularly to treatment and/or prevention of graft versus host disease. The invention provides a method of administration of cpn10 to a donor and/or recipient animal or cells, tissues or organs derived from the donor, although in a particularly advantageous form treatment of both the donor and recipient animal. The method may further include the administration to the donor and/or recipient animal at least one other immunosuppressive agent to prevent or alleviate graft versus host disease.

FIELD OF INVENTION

THIS INVENTION relates to a method of treating graft versus host diseaseand other transplant-related immunological reactions and diseases. Moreparticularly, this invention relates to a method of prophylactic andtherapeutic treatment of graft versus host disease using chaperonin 10.

BACKGROUND OF THE INVENTION

Graft versus host disease (GVHD) is a condition that can develop whenimmunologically-competent cells have been introduced into an individual,for example during bone marrow or stem cell transplantation. GVHD refersto the immunological process whereby the newly transplanted cells mounta rejection response against host tissue. GVHD can develop after thetransplantation or transfusion of bone marrow tissue, haematopoieticstem cells, unirradiated blood products and solid organs containinglymphoid tissue.

There are two types of GVHD, acute and chronic. Acute GVHD developswithin the first three months following transplantation and clinicalsymptoms include dermatitis, enteritis and hepatitis. Chronic GVHDusually develops three months after transplantation and is an autoimmunesyndrome affecting multiple organs and tissues, such as the skin, GItract and liver.

Donor T cells are responsible for triggering the development of GVHD.Donor T cells recognise the host cell antigens as foreign and respond byproliferating and releasing cytokines which in turn may activate cellsof the innate immune system.

Allogeneic bone marrow transplantion or haematopoietic celltransplantation remains the most effective curative therapy for thetreatment of hematological malignancies, such as leukaemia, myeloma,lymphoma and aplastic anaemia. Severe acute GVHD is the primary cause ofmortality and morbidity during bone marrow transplantation. Chronic GVHDcan also result in death and survivors are often severely disabled.

Immunosuppressive drugs play a large part in the prevention, therapeutictreatment and management of acute and chronic GVHD. The drugs may beadministered to the patient before and after the transplant. Currentdrugs used in the therapeutic treatment of GVHD include cyclosporine,methotrexate, tacrolimus, sacrolimus, mycophenolate mofetil andsteroids. Immunosuppression regimens often involve the administration ofa combination of drugs for maximal effect.

Chaperonin 10 (cpn10) is present in a variety of organisms, frombacteria to humans, and is a member of the heat shock family of proteins(chaperones) which are among the most evolutionary stable proteins inexistence. The chaperone molecules are involved in post-translationalfolding, targeting and assembly of other proteins (Hartman et al., 1992,Proc. Natl. Acad. Sci. USA, 89, 3394-8) but do not themselves form partof the final assembled structure (Ellis et al., 1991, Annu. Rev.Biochem. 60, 32147). These proteins play essential roles in normal cellsbut their production is upregulated during cellular stress (eg.metabolic disruption, infection, inflammation, transformation).

It was unexpectedly discovered that chaperonin 10 has the same aminoacid sequence as Early Pregnancy Factor (EPF) (Morton et al.,International Publication WO 95/15338). EPF is a pregnancy-associatedsubstance that appears in the maternal serum within 6-24 hr offertilization (Morton et al., 1974, Nature, 249; 459-460 and Morton etal., 1976, Proc. R. Soc. Lond., 193; 413-9). It is present for at leastthe first half of pregnancy and is essential for continued embryonicgrowth and survival (Morton et al., 1987, Current Topics inDevelopmental Biology 23; 73-92). It is now clear that EPF has manyphysiological functions and its production is not confined to pregnancy.

It has been reported that EPF can act as an immunosuppressant, releasesuppressor factors from lymphocytes (Rolfe et al., 1988, Clin. Exp.Immunol. 73, 219-225) and augment the rosette-inhibiting properties ofan immunosuppressive anti-lymphocyte serum (Morton et al., 1974 and1976, supra). EPF can suppress the delayed-type hypersensitivityreaction to trinitrochlorobenzene in mice (Noonan et al., 1979, Nature,278, 649-51), suppress mitogen-induced lymphocyte proliferation(Athanasas-Platsis, 1993, PhD Thesis, The University of Queensland) andsuppress IFN-γ production by CD4+ T cells.

However, there has been no direct evidence as to whether EPF or cpn10may have potential as an immunosuppressive agent in transplantation, andin particular in the prevention of GVHD. Chaperonin 60, a related heatshock protein, which can also act as an immunosuppressant, has not beenshown to possess any therapeutic effects in GVHD. In fact, the prior artteaches that heat shock proteins may have adverse effects ontransplantation (Ogita et al., 2000, Transplantation, 69,2273-2277).

OBJECT OF THE INVENTION

The present inventors have realized the immunosuppressive drugscurrently used for the therapeutic treatment and management of GVHD havethe following significant short-comings:

-   -   (i) they induce severe side effects, for example, hypertension        which may require additional medication for control,        nephrotoxicity which occurs in up to 40% of patients and        frequently forces the doctor to administer sub-optimal doses of        the drug to limit the toxicity, CNS effects such as tremor,        headache, depression, paraesthesia, blurred vision and seizures,        increased risk of bacterial, fungal or viral infections,        increased risk of cancer, particularly skin cancer, loss of        appetite, nausea and increased hair growth;    -   (ii) GVHD is resistant to the drugs in a significant percentage        of patients and combination drug therapy is required;    -   (iii) the drugs are very expensive; and    -   (iv) the drugs have demonstrated adverse interactions with other        therapeutic drugs, such as antibiotics, NSAIDs, anti-epileptics,        and antifungals, immunizations, such as rubella and polio, and        natural food, such as grapefruit (in the case of cyclosporin).

Therefore there is an enormous demand for the development of a new drugto treat and manage GVHD that has fewer side effects side effects thanthe treatments currently available and is more efficacious in patientsthat show a resistance to the current drugs on the market.

The present inventors have unexpectedly discovered that cpn10 possessesenormous clinical potential as a new therapy in the treatment andmanagement of GVHD.

SUMMARY OF INVENTION

The invention is broadly directed to the use of cpn10 in transplantationand particularly to treatment and/or prevention of graft versus hostdisease.

The invention in a broad form provides administration of cpn10 to adonor and/or recipient animal or cells, tissues or organs derived fromthe donor, although in a particularly advantageous form the inventionprovides treatment of both the donor and recipient animal.

Therefore in a first aspect, the invention provides a method oftherapeutically or prophylactically treating graft versus host disease(GVHD), including the steps of:

-   -   (i) administering a pharmaceutically-effective amount of        chaperonin 10 (cpn10) or a derivative of cpn10 to a donor animal        or cell, organ or tissue obtained therefrom; and    -   (ii) administering to a recipient animal a        pharmaceutically-effective amount of cpn10 or a derivative of        cpn10, to thereby delay, ameliorate, suppress or otherwise        reduce one or more symptoms of GVHD following transplantation of        the one or more cells, tissues or organs to the recipient        animal.

Preferably, the pharmaceutically-effective amount of cpn10 or aderivative of cpn10 is administered to a recipient animal both beforeand after step (ii).

Preferably, the pharmaceutically-effective amount of cpn10 or derivativeof cpn10 administered to an animal is within the range 0.1-100 mg perkg/body weight. More preferably, it is within the range 0.1-10 mg perkg/body weight.

Preferably, the animal is a mammal.

Preferably, the mammal is a human.

Suitably, the cell, tissue or organ is bone marrow or is derived frombone marrow.

Suitably, the method of therapeutically or prophylactically treatingGVHD further includes the step of administering to said donor animaland/or said recipient animal at least one other immunosuppressive agentselected from the group consisting of cyclosporin, tacrolimus,sirolimus, mycophenolate mofetil and methotrexate.

Suitably, the method of therapeutically or prophylactically treatingGVHD further includes the step of administering to said donor animaland/or said recipient animal a steroid.

In a second aspect, there is provided a method of inhibiting,suppressing or otherwise reducing INFα production in an animal includingthe step of administering to said animal a pharmaceutically-effectiveamount of cpn10 or derivative of cpn10 to thereby, inhibit, suppress orotherwise reduce production of TNFα in said animal.

Preferably, the animal is a mammal.

Preferably, the mammal is a human.

According to this aspect, the invention also provides a method ofinhibiting, suppressing or otherwise reducing TNFα production by one ormore cells, tissues or organs obtained from an animal including the stepof administering to said cells, tissues or organs apharmaceutically-effective amount of cpn10 or derivative of cpn10 tothereby inhibit production of INFα by said animal.

In a third aspect, the invention provides a method of inducing,augmenting or otherwise increasing IL-10 production in an animalincluding the step of administering to said animal apharmaceutically-effective amount of cpn10 or derivative of cpn10 tothereby induce, augment or otherwise increase production of IL-10 insaid animal.

Preferably, the animal is a mammal.

Preferably, the mammal is a human.

According to this aspect, the invention also provides a method ofinducing, augmenting or otherwise increasing TNFα production by one ormore cells, tissues or organs obtained from an animal including the stepof administering to said cells, tissues or organs apharmaceutically-effective amount of cpn10 or derivative of cpn10 tothereby induce production of IL-10 by said animal.

In a fourth aspect, there is provided a pharmaceutical composition foruse according to the method of any of the aforementioned aspectscomprising a pharmaceutically-effective amount of cpn10 or a derivativeof cpn10, and a pharmaceutically-acceptable carrier, excipient ordiluent.

Preferably, the at least one other immunosuppressive agent is animmunosuppressive drug or a specific antibody directed against B or Tlymphocytes or surface receptors that mediate their activation.

Preferably, the immunosuppressive drug is any one of cyclosporin,tacrolimus, sirolimus, mycophenolate mofetil and methotrexate.

In a fifth aspect, there is provided a pharmaceutical composition of thefourth aspect further comprising a steroid.

Preferably, said cpn10 protein has an amino acid sequence set forth inFIG. 1 (SEQ ID NO: 1).

Throughout this specification, “comprise”, “comprises” and “comprising”are used inclusively rather than exclusively, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The amino acid sequence of the cpn10 protein (SEQ ID NO:1).

FIG. 2A-G: The effect of in vivo cpn10 treatment on LPS- andalloantigen-induced proinflammatory responses of mice peritonealmacrophages and on T cell differentiation.

FIGS. 3A and B: Survival of mice after bone marrow transplantation andpost-transplant treatment of cpn10. In the post transplant period (day 0to 21) animals were injected subcutaneously with either vehicle(syngeneic, n=8 and allogeneic control group, n=10) or cpn10 (10 and 100μg/animal/day: cpn10 10 μg/day allogeneic, n=10 and cpn10 100 μg/dayallogeneic, n=10). B. Mice GVHD clinical scores plotted over time (0-45days; ** P<0.01).

FIGS. 4A and B: Survival of mice after bone marrow transplantation andpre-transplant treatment of cpn10. Recipient and donor mice were treatedfor 5 days pre-transplant with subcutaneous injections of cpn10 (100μg/day) or control diluent. Five groups of animals were then formed:Group 1: Syngeneic control (n=8) represented B6D2F1 transplanted withsyngeneic B6D2F1 bone marrow and T cells; Group 2: Allogeneic control(n=10) consisted of diluent pre-treated B6D2F1 recipients transplantedwith cells from diluent pre-treated B6 donors; Group 3: Allogeneicrecipient pre-treated (n=10), recipient B6D2F1 mice were treatedpre-transplant with cpn10 and were transplanted with vehicle pre-treatedB6 donor cells; Group 4: Recipient B6D2F1 mice pretreated with diluentonly that received transplant from cpn10 pre-treated B6 donors(allogeneic: donor pretreated, n=10); and Group 5: Both B6D2F1recipients and B6 donor mice were pretreated with cpn10 prior totransplantation (allogeneic: recipient and donor pre-treated, n=10).(*P<0.01 versus allogeneic control). B. Mice GVHD clinical scoresplotted over time (0-30 days; *** P<0.001).

DETAILED DESCRIPTION OF INVENTION

The inventors have demonstrated that cpn10 has significantimmunosuppressive activity in an in vivo mouse transplantation model andthat cpn10 treatment increases the survival rate of mice suffering fromGVHD. This is the first demonstration of the beneficialimmunosuppressive effects of cpn10 and increased survival rates in an invivo GVHD model.

The effectiveness of the cpn10 treatment is increased if both donor andrecipient animals are treated with cpn10 prior to the transplantprocedure. The invention also demonstrates that cpn10 inhibitslipopolysaccharide-mediated TNFα secretion and promotes IL-10 productionin mouse macrophages. IL 10 is a potent immunosuppressive cytokine thatis a powerful inhibitor of adaptive and innate immune responses to LPS.

Acute GVHD following allogeneic bone marrow transplantation (BMT) is a Tcell mediated disease in which donor T cells recognise disparate hostantigens and differentiate in a Th1 dominant fashion. The resulting Tcell derived Th1 cytokines prime donor mononuclear cells that releasecytopathic quantities of inflammatory cytokines (e.g. TNFα) when theycome into contact with lipopolysaccharide (LPS). LPS leaks through thegastrointestinal mucosa which is damaged by GVHD and by the precedingradiation. Therefore TNFα, together with the dysregulated cytotoxiccytokine production induces apoptosis in host tissue. GVHD mortality inBMT models is prevented by T cell directed immunosuppression,particularly by agents that inhibit IL-2 generation.

The present invention is exemplified in respect of bone marrowtransplantation. However, it will be appreciated the concept isapplicable to other cells, tissues and organs that includeimmuno-competent cells capable of initiating an immune response in thehost. Non-limiting examples of such cells, tissues and organs includeliver, lung, heart, kidney and stem and progenitor cells.

The invention as described herein may be broadly applicable to anyanimal but is particularly directed to mammals, and preferably humans.For example, the invention may be directed to a transplantation inlivestock, domestic animals, laboratory animals and performance animals(for example, racehorses and camels).

For the purposes of this invention, by “isolated” is meant material thathas been removed from its natal state or otherwise been subjected tohuman manipulation. Isolated material may be substantially oressentially free from components that normally accompany it in itsnatural state, or may be manipulated so as to be in an artificial statetogether with components that normally accompany it in its naturalstate. Isolated material may be in native, chemical synthetic orrecombinant form.

By “protein” is meant an amino acid polymer. The amino acids may benatural or non-natural amino acids D- and L-amino acids, as are wellunderstood in the art.

A “peptide” is a protein having no more than fifty (50) amino acids.

A “polypeptide” is a protein having more than fifty (50) amino acids.

The term “nucleic acid” as used herein designates single-ordouble-stranded mRNA, RNA, cRNA, RNAi and DNA inclusive of cDNA andgenomic DNA.

By “immunosuppressive agent” is meant an agent that can prophylacticallyor therapeutically suppress an autoimmune or immune response against atransplanted allogeneic or xenogeneic cell, tissue or organ, or tosuppress graft versus host disease.

Preferably, the pharmaceutically effective amount of cpn10 administeredto an individual is within the range 0.1-100 mg.

More preferably, the pharmaceutically-effective amount of cpn10administered to an individual is within the range 0.1-10 mg.

It will be appreciated by the skilled person that the aforementionedpharmaceutically-effective amounts are calculated in terms of a typical70 kg human. Accordingly, doses may vary depending on the weight, age,sex, general health and fitness of the individual and any othertreatments to which the individual is being subjected. Furthermore, theamount of cpn10 administered will be interdependent with the frequencyand timing of administration.

It will also be appreciated that the aforementionedpharmaceutically-effective amounts of cpn10 can be administered toanimals, for example, domestic animals and livestock. Doses would varydepending on the weight and type of animal, as would be apparent tothose of skill in the art.

The cpn10 administered to a human or other animal may be any form ofisolated cpn10, including but not limited to recombinant cpn10 (SEQ IDNO: 1), native cpn10, pegylated cpn10, recombinant cpn10-GSM or anyother derivative protein of cpn10.

Suitable cpn10 nucleotide and amino acid sequences are well known in theart, although for convenience the skilled person is referred to thefollowing mammalian cpn10 sequences:

-   -   (i) human cpn10 (NCBI Entrez Accession No. U07550; Chen et al.,        1994, Biochim. Biophys. Acta, 1219, 189-190)    -   (ii) mouse cpn10 (NCBI Entrez Accession No. U09659; Dickson et        al, 1994, J. Biol. Chem., 269, 26858-864); and    -   (iii) rat cpn10 (NCBI Entrez Accession No. X71429; Ryan et al.,        1994, FEBS Lett, 337, 152-156).

Both donor and recipient can be treated with cpn10 prior to thetransplant procedure.

Preferably, the donor undergoes cpn10 treatment for no more than 7 daysprior to the transplant procedure. More preferably, the donor undergoescpn10 treatment for 2 to 5 days prior to the transplant procedure.

Preferably, the recipient undergoes cpn10 treatment for no more than 7days prior to the transplant procedure and no more than 90 days afterthe procedure. More preferably, the recipient undergoes cpn10 treatmentfor 2 to 5 days prior to the transplant procedure and no more than 60days after the procedure. Even more preferably, the recipient undergoescpn10 treatment for 2 to 5 days prior to the transplant procedure and 10to 30 days after the procedure.

As used herein, “derivative” proteins of the invention are proteins,such as cpn10 proteins, which have been altered, for example byconjugation or complexing with other chemical moieties or bypost-translational modification techniques as would be understood in theart, inclusive of fusion partner proteins.

Other derivatives contemplated by the invention include, but are notlimited to, pegylation, modification to side chains, incorporation ofunnatural amino acids and/or their derivatives during peptide,polypeptide or protein synthesis and the use of crosslinkers and othermethods which impose conformational constraints on the polypeptides,fragments and variants of the invention. Examples of side chainmodifications contemplated by the present invention includemodifications of amino groups such as by acylation with aceticanhydride; acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; amidination with methylacetimidate;carbamoylation of amino groups with cyanate; pyridoxylation of lysinewith pyridoxal-5-phosphate followed by reduction with NaBH₄; reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; and trinitrobenzylation of amino groups with2,4,6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodimide activation viaO-acylisourea formation followed by subsequent derivitization, by way ofexample, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation ofheterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acidoxidation to cysteic acid; formation of mercurial derivatives using4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;2-chloromercuri-4-nitrophenol, phenylmercury chloride, and othermercurials; formation of a mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; carboxymethylation with iodoacetic acid or iodoacetamide; andcarbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of theindole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides orby oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified byN-carbethoxylation with diethylpyrocarbonate or by alkylation withiodoacetic acid derivatives.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include but are not limited to, use of 4-amino butyricacid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine,norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/orD-isomers of amino acids.

Derivatives may also include fusion partners and epitope tags. Wellknown examples of fusion partners include, but are not limited to,glutathione-S-transferase (GST), Fc portion of human IgG, maltosebinding protein (MBP) and hexahistidine (HIS₆) (SEQ ID NO: 2), which areparticularly useful for isolation of the fusion protein by affinitychromatography. For the purposes of fusion polypeptide purification byaffinity chromatography, relevant matrices for affinity chromatographyare glutathione-, amylase-, and nickel- or cobalt-conjugated resinsrespectively. Many such matrices are available in “kit” form, such asthe QIAexpress™ system (Qiagen) useful with (HIS₆) (SEQ ID NO: 2) fusionpartners and the Pharmacia GST purification system.

One particular example of a fusion partner is GST, such as described inRyan et al. (supra). In some cases, the fusion partners also haveprotease cleavage sites, such as for Factor X_(a) or Thrombin, whichallow the relevant protease to partially digest the fusion polypeptideof the invention and thereby liberate the recombinant polypeptide of theinvention therefrom. The liberated polypeptide can then be isolated fromthe fusion partner by subsequent chromatographic separation. Uponcleavage of GST-cpn10 the derivative GSM-cpn10 protein is produced, forexample.

Fusion partners according to the invention also include within theirscope “epitope tags”, which are usually short peptide sequences forwhich a specific antibody is available. Well known examples of epitopetags for which specific monoclonal antibodies are readily availableinclude c-myc, haemagglutinin and FLAG tags.

Cpn10 proteins of the invention (inclusive of fragments, variants,derivatives and homologues) may be prepared by any suitable procedureknown to those of skill in the art, including chemical synthesis andrecombinant expression.

Preferably, cpn10 is recombinant cpn10.

For example, the recombinant cpn10 protein may be prepared by aprocedure including the steps of:

-   -   (i) preparing an expression construct which comprises an        isolated nucleic acid encoding cpn10, operably-linked to one or        more regulatory nucleotide sequences in an expression vector;    -   (ii) transfecting or transforming a suitable host cell with the        expression construct; and    -   (iii) expressing the recombinant protein in said host cell.

An “expression vector” may be either a self-replicatingextra-chromosomal vector such as a plasmid, or a vector that integratesinto a host genome.

By “operably-linked” is meant that said regulatory nucleotidesequence(s) is/are positioned relative to the recombinant nucleic acidof the invention to initiate, regulate or otherwise controltranscription.

Regulatory nucleotide sequences will generally be appropriate for thehost cell used for expression. Numerous types of appropriate expressionvectors and suitable regulatory sequences are known in the art for avariety of host cells. Typically, said one or more regulatory nucleotidesequences may include, but are not limited to, promoter sequences,leader or signal sequences, ribosomal binding sites, transcriptionalstart and termination sequences, translational start and terminationsequences, splice donor/acceptor sequences and enhancer or activatorsequences.

Constitutive or inducible promoters as known in the art are contemplatedby the invention and include, for example, tetracycline-repressible andmetallothionin-inducible promoters. The promoters may be eithernaturally occurring promoters, or hybrid promoters that combine elementsof more than one promoter.

In a preferred embodiment, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectablemarker genes are well known in the art and will vary with the host cellused.

Suitable host cells for expression may be prokaryotic or eukaryotic,such as Escherichia coli (DH5α for example), yeast cells, SF9 cellsutilized with a baculovirus expression system, CHO cells, COS, CV-1 and293 cells, without limitation thereto.

The recombinant cpn10 protein may be conveniently prepared by a personskilled in the art using standard protocols as for example described inSambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold SpringHarbor Press, 1989), incorporated herein by reference, in particularSections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubelet al., (John Wiley & Sons, Inc. 1995-1999), incorporated herein byreference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS INPROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995-1999)which is incorporated by reference herein, in particular Chapters 1, 5and 6.

An example of production and purification of recombinant synthetic cpn10using the pGEX system is provided in WO 95/15338. A high yieldingbacterial expression system known to produce active cpn10 (Ryan et al.,supra) was used to produce the cpn10 (SEQ ID NO: 1) used in theexperiments described herein.

Pharmaceutical Compositions

The invention provides a use of cpn10 for the therapeutic treatment ofdiseases or medical conditions caused by cell, tissue or organtransplantation, in particular GVHD.

The invention also provides pharmaceutical compositions that comprisecpn10 or a derivative of cpn10.

Suitably, the pharmaceutical composition comprises an appropriatepharmaceutically-acceptable carrier, diluent or excipient.

Suitably, the pharmaceutical composition comprises cpn10 or a derivativeof cpn10, a pharmaceutically-acceptable carrier, diluent or excipientand at least one other immunosuppressive agent. Preferably, the otherimmunosuppressive agent is an immunosuppressive drug or a specificantibody directed against B or T lymphocytes or surface receptors thatmediate their activation. More preferably, the immunosuppressive agentis any one of cyclosporin, tacrolimus, sirolimus, mycophenolate mofetiland methotrexate. The pharmaceutical composition may also comprise asteroid.

By “pharmaceutically-acceptable carrier, diluent or excipient” is meanta solid or liquid filler, diluent or encapsulating substance that may besafely used in systemic administration. Depending upon the particularroute of administration, a variety of carriers, well known in the artmay be used. These carriers may be selected from a group includingsugars, starches, cellulose and its derivatives, malt, gelatine, talc,calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline and saltssuch as mineral acid salts including hydrochlorides, bromides andsulfates, organic acids such as acetates, propionates and malonates andpyrogen-free water.

A useful reference describing pharmaceutically acceptable carriers,diluents and excipients is Remington's Pharmaceutical Sciences (MackPublishing Co. N.J. USA, 1991) which is incorporated herein byreference.

Any safe route of administration may be employed for providing a patientwith the composition of the invention. For example, oral, rectal,parenteral, sublingual, buccal, intravenous, intra-articular,intramuscular, intra-dermal, subcutaneous, inhalational, intraocular,intraperitoneal, intracerebroventricular, transdermal and the like maybe employed. Intra-muscular and subcutaneous injection is appropriate,for example, for administration of immunogenic compositions, vaccinesand DNA vaccines.

Dosage forms include tablets, dispersions, suspensions, injections,solutions, syrups, troches, capsules, suppositories, aerosols,transdermal patches and the like. These dosage forms may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of the therapeuticagent may be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, the controlled releasemay be effected by using other polymer matrices, liposomes and/ormicrospheres.

The above compositions may be administered in a manner compatible withthe dosage formulation, and in such amount as ispharmaceutically-effective. The dose administered to a patient, in thecontext of the present invention, should be sufficient to affect abeneficial response in a patient over an appropriate period of time. Thequantity of agent(s) to be administered may depend on the subject to betreated inclusive of the age, sex, weight and general health conditionthereof, factors that will depend on the judgement of the practitioner.

So that the present invention may be more readily understood and putinto practical effect, the skilled person is referred to the followingnon-limiting examples.

EXAMPLES

Methods

Transplantation

Mice were transplanted according to a standard protocol as described inHill et al., 1997, Blood, 90, 3204-3213, and Hill et al., 1999, J. Clin.Invest., 104, 459-467. On day 0 B6D2F1 mice received 1400 cGy total bodyirradiation (TBI, ¹³⁷CS source) in two doses separated by three hours tominimize gastrointestinal toxicity. 5×10⁶ bone marrow cells and 2×10⁶nylon wool purified splenic donor T cells from B6 mice (allogenic) orB6D2 μl mice (syngeneic) were resuspended in 0.25 ml of Leibovitz's L-15media and injected intravenously into the irradiated recipients.

Preparation of Recombinant cpn10

XL1-Blue E. coli cells were transformed with cpn10 using the expressionvector pPL550 and grown at 37° C. Cells in exponential growth wereinduced to express protein by temperature increase to 42° C. for 4 h.Cells were pelleted, resuspended in 30 ml 0.025 M TrisHCl pH 8.0 andstored at −30° C.

A cell pellet from a 1 L culture was thawed, cells were lysed withlysozyme (100 μg/ml; 15 min at 37° C.), followed by sonication (5×10sec, 4° C.) and cellular debris was removed by centrifugation (30 min,4° C., 48 384×g).

Cpn10 was purified from the clarified lysate by ion-exchange andhydrophobic interaction chromatography. The protein was identified incolumn fractions as an ˜10 kDa band using SDS-PAGE on 10-20%Tris-Tricine gels (100×100×1 mm; Novex).

Lysate was applied to a 200 ml column of Macroprep HighQ (BIO-RAD) using0.025 M TrisHCl pH 8.0 as running buffer at a flow rate of 8 ml/min. Theunbound fraction was retained and pH adjusted to 6.8. The sample wasapplied to a 5 ml EconoPac S cartridge (BIO-RAD) using 0.025 M sodiumphosphate buffer pH 6.8 as running buffer at a flow rate of 2 ml/min Thecolumn was eluted with a gradient of 0→1 M NaCl in 0.025 M sodiumphosphate buffer pH 6.8, applied over 30 min at 2 ml/min

Cpn10 containing fractions were pooled and an equal volume of 3 M(NH₄)₂SO₄ in 0.05 M sodium phosphate buffer pH 6.8 was added. The samplewas applied to a 5 ml Econo-pac Methyl HIC cartridge using 1.5 M(NH₄)₂SO₄ in 0.05 M sodium phosphate buffer pH 6.8 as running buffer ata flow rate of 2 ml/min. The column was eluted with a gradient of 1.5→0M (NH₄)₂SO₄ in 0.05 M sodium phosphate buffer pH 6.8, applied over 15min at 2 ml/min.

Cpn10 containing fractions were pooled, dialysed against salineovernight, dispensed in appropriate aliquots and stored at −30° C.

Cpn10 Treatment

Recombinant human cpn10 was diluted in PBS before injection. Mice wereinjected subcutaneously with cpn10 each day (10 μg/dose or 100 μg/dose)before or after BMT as described. Mice from the control groups receivedinjection of diluent only.

Assessment of GVHD

The degree of systemic GVHD was assessed by survival and by a scoringsystem which sums changes in five clinical parameters: weight loss,posture (hunching), activity, fur texture and skin integrity (maximumindex=10) (Cooke et al., 1996, Blood, 88, 3230-3239; Hill et al., 1999,J. Clin. Invest., 104, 459-467). Individual mice were ear-tagged andgraded weekly from 0 to 2 for each criterion. Animals with severeclinical GVHD (scores>6) were sacrificed according to ethical guidelinesand the day of death deemed to be the following day.

Statistical Analysis

Survival curves were plotted using Kaplan-Meier estimates and comparedby log-rank analysis. The Mann Whitney-U Test was used for thestatistical analysis of clinical scores. P<0.05 was consideredstatistically different

Example 1 In Vitro Mouse Macrophage Experiments

In vitro experiments were carried out to determine the effect of cpn10in a physiological cell population.

Mice

Female C57BL/6 (B6, H-2^(b), Ly-5.2⁺), B6 Ptprc^(a) Ly-5^(a) (H-2^(b),Ly-5.1⁺) and B6D2F1 (H-2^(b/d), Ly-5.2⁺) mice were purchased from theAustralian Research Centre (Perth, Western Australia, Australia).C57BL/6 IL-10^(−/−) mice (B6, H-2^(b), Ly-5.2⁺) were supplied by theAustralian National University (Canberra, Australia). The age of miceused as transplant recipients ranged between 8 and 14 weeks. Mice werehoused in sterilised micro-isolator cages and received acidifiedautoclaved water (pH 2.5) and normal chow for the first two weekspost-transplantation.

Bone Marrow Transplantation

Mice were transplanted according to a standard protocol (Hill et al,1997 supra). Briefly, on day 1, B6D2F1 mice received 1300 cGy total bodyirradiation (¹³⁷Cs source at 108 cGy/min), split into two dosesseparated by 3 hours to minimize gastrointestinal toxicity. Donor bonemarrow (5×10⁶ per animal) and splenic T cells (3×10⁶ per animal) wereresuspended in 0.25 ml of Leibovitz's L-15 media (Gibco BRL,Gaithersburg Md.) and were injected intravenously into recipients.Survival was monitored daily, and the GVHD clinical scores were measuredweekly. Cpn10 or control diluent was injected subcutaneously at doses of100 ug per animal. The degree of systemic GVHD was assessed as describedabove.

Cell Cultures

The culture media used throughout was 10% FCS/IMDM (JRH Biosciences,Lenexa, Kans.) supplemented with 50 units/ml penicillin, 50 μg/mlstreptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mMnon-essential amino acid, 0.02 mM β-mercaptoethanol, and 10 mM HEPES.Experiments were performed at pH 7.75 and 37° C. in a humidifiedincubator supplemented with 5% CO₂.

For in vitro LPS stimulation experiments, peritoneal macrophages orsplenocytes were stimulated with graded concentrations of LPS, TNFα andIL-10 determined in culture supernatants at 5 hours and 48 hoursrespectively. For in vitro allo-antigen experiments, purified C57BL/6 Tcells were cultured in 96 well plates (Becton Dickinson, Franklin Lakes,N.J.) with 10⁵ irradiated (2000 cGy) B6D2 μl peritoneal macrophagesprimary MLC) and supernatants harvested at 72 hours. Cultures were thenpulsed with ³H-thymidine (1 μCi per well) and proliferation wasdetermined 16 hrs later on a 1205 Betaplate reader (Wallac, Turku,Finland). For in vitro mitogen stimulation, purified C57BL/6 T cellswere cultured in flat bottomed 96 well plates, pre-coated withmonoclonal CD3 and CD28 at final concentrations of 10 μg/ml.Supernatants were harvested at 48 hours and cultures pulsed with³H-thymidine (1 μCi per well). Proliferation was determined 16 hrslater.

Cytokine ELISAS

The antibodies used in the IFNγ, I-10, IL-4 and TNFα assays werepurchased from PharMingen (San Diego, Calif.) and assays were performedaccording to the manufacturer's protocol. Briefly, samples were diluted1:3 to 1:24 and cytokines were captured by the specific primarymonoclonal antibody (mAb) and detected by biotin-labelled secondarymAbs. The biotin-labelled assays were developed with strepavidin andsubstrate (Kirkegaard and Perry laboratories, Gaithersburg, Md.). Plateswere read at 450 nm using the Spectraflour Plus microplate reader(Tecan, Durham, N.C.). Recombinant cytokines (PharMingen) were used asstandards for ELISA assays. Samples and standards were run in duplicateand the sensitivity of the assays were 0.063 U/ml for IFNγ, and 15 pg/mlfor IL-10, IL-4 and TNFα.Results

In vivo administration of cpn10 reduced the capacity of peritonealmacrophages to produce TNFα (FIG. 2A). B6 mice (n=3) were treated for 5days with cpn10 (100 μg, once daily) (cpn10+) or control diluent(cpn10−). Peritoneal macrophages were harvested by peritoneal lavage onday 6 and pooled from individual animals within the treatment group.Cells were plated at 2×10⁵/well in the absence (not shown) or presenceof LPS (1 ug/ml). Culture supernatants were collected at 5 hours andlevels of TNFα (pg/ml) were assessed by ELISA. Results are normalized toproduction per 10⁵ macrophages based on CD11b staning. FIG. 2A showsdata from two identical experiments. LPS-induced secretion of TNFα wasreduced by 40% from these cells.

In vivo treatment with cpn10 augmented IL-10 production from splenocytes(FIG. 2B). B6 mice were treated with either cpn10 or control diluent asdescribed above. Splenocytes were harvested on day 6 and pooled fromindividual animals within a treatment group before culture at 5×10⁵/wellin the absence (not shown) or presence of LPS (10 ug/ml). Culturesupernatants were collected at 48 hours and levels of IL-10 (pg/ml)determined by ELISA. The data from two identical experiments is shown inFIG. 2B. Significantly increased IL-10 production was observed comparedto control animals.

In vivo treatment with cpn10 reduced TNFα production from IL10−/−peritoneal macrophages (FIG. 2C). IL10^(−/−) B6 mice were treated withcpn10 or control diluent and peritoneal macrophages were harvested byperitoneal lavage on day 6 and pooled from individual animals within thetreatment group. After 5 hours of cultivation in the absence (LPS 0) orpresence of LPS (0.1, 1 and 10 ug/ml), the amount of TNFα was determinedin the culture supernatants. Cpn10-mediated reduction in LPS-inducedTNFα production (FIG. 2A) does not require IL-10 since similarreductions in TNFα secretion were observed when peritoneal macrophagesfrom cpn10 treated IL-10^(−/−) mice were stimulated with LPS in vitro.Thus reduced TNFα secretion and increased IL-10 production appears to beindependent consequences of cpn10 treatment.

Cpn10 treatment did not appear to affect T cell IFNγ or IL-4 secretion.Previous reports have suggested that cpn10 can inhibit T cellproliferation in response to mitogen (Morton H. 1998 Immunol. CellBiol., 76, 483496). Th1 immune responses are usually characterised byproinflammatory cytokines such as TNFα and IFNγ. Th2 responses involveIL-4 and IL-10 secretion and regulatory T cell (Treg) responses arecharacterised by IL-10 and TGFβ production. Since Th2 and Treg responsessuppress proliferation and Th1 responses suppress cytokine production,the ability of cpn10 to influence T cell differentiation wasinvestigated.

In vivo administration of cpn10 did not affect proliferative response ofT lymphocytes to alloantigen (FIG. 2D). B6 mice (n=3) were treated withdaily injections of cpn10 or control diluent. The splenocyte-derived Tcell populations were stimulated in vitro with allogenic splenocytes for7 days in a mixed lymphocyte culture (MLC). Purified T cells (0.5×10⁵,1×10⁵ and 2×10⁵/well) were stimulated with irradiated allogeneic B6D2F1peritoneal macrophages (0.5×10⁵/well) and proliferation was measured at72 hours via standard ³[H] Thymidine incorporation assay. The valuesplotted in FIG. 2D represent mean±SE of triplicate wells. Theproliferation of T cells within these MLCs did not differ significantlybetween cpn10 treated and control animals indicating that that cpn10 isnot a T cell growth regulator.

FIGS. 2E-G show the effects of in vivo administration of cpn10 on T celldifferentiation. B6 animals were treated in vivo with cpn10 as describedabove. T cells (2×10⁶/well) from cpn10 treated and vehicle treatedanimals were stimulated in culture for 7 days with irradiated allogeneicB6D2F1 splenocytes (3×10⁶/well) (primary mixed lymphocyte culture). Onday 7 T cells were collected and re-stimulated with plate-boundantibodies to CD3 and CD28 which stimulate T cells. Culture supernatantswere harvested at 24 hours and concentrations of IFNγ, IL-4 and IL-10were determined by ELISA. The concentration values in FIGS. 4E-Grepresent the mean±SE of triplicate wells.

Neither T cell IL-4 secretion (FIG. 2E) nor IFNγ secretion (FIG. 2F)differed significantly between cpn10 treated and control animalssuggesting that cpn10 does not influence the Th1/Th2 balance.

In contrast, T cell IL-10 secretion was significantly elevated (FIG.2G). A similar elevation in IL-10 secretion was also observed when cpn10treatment was carried out in vitro during cell culture (MLC), ratherthan in vivo, and proliferation, IFNγ and IL-4 was again unaffected(data not shown). These data indicate that cpn10 can enhance IL-10production in response to stimulation with LPS and alloantigen, andsuggests that cpn10-enhanced IL-10 production may be due in part from Tcell-derived IL-10.

Example 2 Effect of Pre- and Post-transplant Treatment with cpn10 in thein Vivo GVDH Model

In vivo experiments were conducted to investigate if the administrationof cpn10 in the peri-transplant period could prevent GVHD.

Post-treatment Administration of cpn10

Bone marrow cells (5×10⁶/animal) and purified T cells (described above)from donor B6 mice were transplanted into lethally irradiated (1100 cGy)B6D2F1 recipient mice (allogeneic groups). B6D2F1 recipients insyngeneic control group received equal doses of B6D2F1 bone marrow and Tcells. In the post-transplant period (day 0 to 21) animals were injectedsubcutaneously with either vehicle (syngeneic, n=8 and allogeneiccontrol group, n=10) or cpn10 (10 and 100 μg/animal/day: cpn10 10 μg/dayallogeneic, n=10 and cpn10 100 μg/day allogeneic, n=10). GVHD clinicalscores (as described above) were determined as a measure of GVHDseverity in surviving animals. Clinically milder GVHD was observed onday 14 and 21 in allogeneic animals injected with 100 μg/day of cpn10compared to the vehicle treated allogeneic control (**P<0.01). There wasno significant difference in the percent of survival between vehicleinjected and cpn10 injected allogeneic groups. FIG. 3 shows micesurvival curves by Kaplan-Meier analysis.

Administration of cpn10 after bone marrow transplantation (BMT) failedto prevent GVHD mortality and only briefly reduced morbidity asdetermined by GVHD clinical scores (FIG. 3B). Clinically milder GVHD wasobserved on day 14 and day 21 in alogeneic animals injected with 100μg/day cpn10 compared to the vehicle-treated allogeneic control(**P<0.01).

Pretreatment Administration of cpn10

Recipient B6D2F1 and donor B6 mice were treated for 5 dayspre-transplant with either cpn10 (100 μg/day/subcutaneously) or controldiluent. Bone marrow (5×10⁶/animal) and T cells (3×10⁶/animal) harvestedon day 6 from B6 donor mice were transplanted into lethally irradiatedB6D2F1 recipients. Five groups of recipients were formed:

Group 1: Syngeneic control (n=8) represented B6D2F1 transplanted withsyngeneic B6D2F1 bone marrow and T cells.

Group 2: Allogeneic control (n=10) consisted of diluent pre-treatedB6D2F1 recipients transplanted with cells from diluent pre-treated B6donors.

Group 3: Allogeneic recipient pre-treated (n=10), recipient B6D2F1 micewere treated pre-transplant with cpn10 and were transplanted withvehicle pre-treated B6 donor cells.

Group 4: Recipient B6D2F1 mice pretreated with diluent only thatreceived transplant from cpn10 pre-treated B6 donors (allogeneic donorpretreated, n=10).

Group 5: Both B6D2F1 recipients and B6 donor mice were pretreated withcpn10 prior to transplantation (allogeneic recipient and donorpre-treated, n=10).

GVHD clinical scores as described above were determined as a measure ofGVHD severity in surviving animals.

Significantly lower clinical scores were observed on day 7 in Group 5(both recipients and donors pretreated with cpn10 prior totransplantation) compared to the allogeneic control group (FIG. 4B;***P<0.001). FIG. 4 shows mice survival curves by Kaplan-Meier analysis.

The administration of cpn10 to transplant donors and recipients for 5days Prior to transplant significantly delayed GVHD mortality (*P<0.001versus allogeneic control). In addition, the severity of GVHD asdetermined by the clinical score was also reduced early after BMT. Theability of cpn10 to delay GVHD mortality when administered prior to BMTis consistent with the anti-inflammatory effect described here, i.e. alimitation of TNFα production (Hill et al., 1998 J. Clin. Invest., 102,115-123; Hill et at, 1997, supra). The failure of cpn10 administrationafter BMT to prevent the development of GVHD, is consistent with anabsence of an effect on T cell activation and differentiation at thedoses and scheduling used in this Example, as was characterised in FIG.3.

Transplant conditioning (lethal irradiation) in these models sets up aprocess of progressive GI tract injury, LPS leak and inflammatorycytokine generation which in turn induces further GI tract injury and sothe process continues as a positive feedback loop. The process may beinterrupted by pharmacological agents that protect the gut fromradiation injury (such as IL-11 and Keratinocyte Growth Factor;Krijanovski et al., 1999, Blood, 94, 825-831), direct LPS antagonists(Cooke et al., 2001, J. Clin. Invest., 107, 1581-1589), or inhibitors ofTNFα itself (Hill et al., 1997 supra). However, these agents tend todelay rather than prevent GVHD unless TNFα is neutralised completely, orthere are additional effects on T cell activation and differentiation.Thus the delay in GVHD mortality by the administration of cpn10 isconsistent with a limitation of LPS signalling and subsequent TNFαproduction early after BMT but also a failure to impact on subsequentalloreactive T cell function.

Therefore cpn10 has the potential to become an important therapeuticdrug in the treatment of GVHD. The increased effectiveness of treatmentof GVHD observed when both donor and recipient are administered withcpn10 prior to the transplant procedure is consistent with the fact thatTNFα originates from both tissue or organ sources in the post transplantperiod (Cooke et al., 2000, J. Immunol., 165, 6612-6619; Speiser et al.,1997, J. Immunol., 158, 5185-5190).

Furthermore, cpn10 may be useful in the treatment of GVHD in combinationwith traditional immunosuppressive agents that limit the secondaryadaptive immune response.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. It will therefore beappreciated by those of skill in the art that, in light of the instantdisclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention.

All computer programs, algorithms, patent and scientific literaturereferred to herein is incorporated herein by reference.

1. A pharmaceutical composition comprising: a pharmaceutically-effectiveamount of cpn10 comprising the amino acid sequence set forth in FIG. 1(SEQ ID NO:1), and a pharmaceutically-acceptable carrier, excipient ordiluent.
 2. An isolated polypeptide comprising the amino acid sequenceset forth in FIG. 1 (SEQ ID NO:1).